Xylose utilizing zymomonas mobilis with improved ethanol production in biomass hydrolysate medium

ABSTRACT

Xylose-utilizing, ethanol producing strains of  Zymomonas mobilis  with improved performance in medium comprising biomass hydrolysate were isolated using an adaptation process. Independently isolated strains were found to have independent mutations in the same coding region. Mutation in this coding may be engineered to confer the improved phenotype.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with United States Government support underAward No. DE-FC36-07GO17056 awarded by the Department of Energy. TheU.S. Government has certain rights in this invention.

FIELD OF THE INVENTION

The invention relates to the fields of microbiology and fermentation.More specifically, mutant Zymomonas strains with improved growth andethanol production in biomass hydrolysate medium were isolated andcharacterized.

BACKGROUND OF THE INVENTION

Production of ethanol by microorganisms provides an alternative energysource to fossil fuels and is therefore an important area of currentresearch. It is desirable that microorganisms producing ethanol, as wellas other useful products, be capable of growing and producing ethanol ina medium that does not impact the human food supply, such as avoidinguse of sugars produced from corn grain. As a result of developments incellulosic biomass processing, glucose, xylose, and other sugars may bereleased in high concentrations in a biomass hydrolysate for use infermentation. As such, conversion of biomass to ethanol poses greatpossibility for improving environmental impacts by using renewablenon-food resources to provide an alternative to fossil fuels.

Zymomonas mobilis and other bacterial ethanologens which do notnaturally utilize xylose have been genetically engineered for xyloseutilization to improve growth and ethanol production by using more ofthe sugars in biomass hydrolysate. However, growth and ethanolproduction in biomass-hydrolysate containing medium is typically notoptimal due to the presence of acetate and other compounds that areinhibitory to microorganisms. Disclosed in commonly owned and co-pendingUnited States Patent Publication US20110014670A1 is a method forproducing an improved xylose-utilizing Zymomonas strain that is moretolerant to acetate and ethanol in the medium, as well as strainsisolated by the method.

The toxic effect of single compounds likely to be found in thehydrolysates of pretreated biomass is described in Delegenes et al.((1996) Enzymes and Microbial Technology 19:220-224). Adaptation ofxylose-fermenting Zymomonas mobilis to conditioned dilute acid yellowpoplar hemicellulose hydrolysate is described in Lawford et al. ((1999),Applied Biochemistry and Biotechnology 77:191-204).

There remains a need for isolated xylose-utilizing Zymomonas ethanologenstrains with improved ethanol production during fermentation in biomasshydrolysate medium, and methods for genetic engineering to produceimproved strains.

SUMMARY OF THE INVENTION

The invention provides recombinant xylose-utilizing Zymomonas strainswith improved growth and ethanol production in biomass hydrolysatemedium. In addition, the invention provides methods of making improvedZymomonas strains for use in hydrolysate medium and methods of makingethanol using said strains.

Accordingly, the invention provides a recombinant, xylose-utilizing,ethanol-producing microorganism of the genus Zymomonas, having at leastone genetic modification in the zmo1432 open reading frame.

In one aspect the invention provides a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO: 3 and SEQ IDNO: 4, and polynucleotides encoding the same.

In another aspect the invention provides a method for the production ofa recombinant Zymonomas ethanologen comprising:

-   -   a) providing a xylose-utilizing, ethanol-producing microorganism        of the genus Zymomonas;    -   b) providing a polynucleotide encoding a protein having the        amino acid sequence selected from the group consisting of: SEQ        ID NO:3 and SEQ ID NO:4; and    -   c) introducing the polynucleotide of b) into the microorganism        of a);    -   wherein the endogenous zmo1432 coding region is disrupted.

In an alternate aspect the invention provides a method for theproduction of a recombinant Zymonomas ethanologen comprising:

-   -   a) providing a xylose-utilizing, ethanol-producing microorganism        of the genus Zymomonas comprising a zmo1432 open reading frame        encoding a polypeptide having the amino acid sequence as set        forth in SEQ ID NO: 2; and    -   b) introducing a mutation in the zmo1432 open reading frame        of a) such that expression of the mutated open reading frame        expresses a polypeptide having an amino acid sequence selected        from the group consisting of SEQ ID NO:3 and SEQ ID NO:4.

In another aspect the invention provides a method for the production ofethanol comprising:

-   -   a) providing the recombinant Zymomonas of the invention;    -   b) providing a biomass hydrolysate medium comprising xylose; and    -   c) growing the Zymomonas of a) in the biomass hydrolysate medium        of b) wherein ethanol is produced.

BRIEF DESCRIPTION OF THE FIGURES, BIOLOGICAL DEPOSITS AND SEQUENCEDESCRIPTIONS

Applicants have made the following biological deposits under the termsof the Budapest Treaty on the International Recognition of the Depositof Microorganisms for the Purposes of Patent Procedure:

Information on Deposited Strains

Depositor Identification International Depository Reference DesignationDate of Deposit Zymomonas ZW658 ATCC No PTA-7858 Sep. 12, 2006

FIG. 1 shows a graph of fermentation of corn cob hydrolysate over time,comparing glucose and xylose utilization and ethanol production forZymomonas strains and Adapted 7-31.

FIG. 2 shows a graph of fermentation of corn cob hydrolysate over time,comparing glucose and xylose utilization and ethanol production forZymomonas strains ZW705, Adapted 7-31, and Adapted 5-6.

FIG. 3 shows an alignment of the protein encoded by zmo1432 and theBcemn03_(—)1426 protein of Burkholderia cenocepacia.

FIG. 4 shows an alignment of the protein encoded by zmo1432 and the E.coli AaeB protein.

The following sequences conform with 37 C.F.R. §§1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (2009) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions. The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID NO:1 is the nucleotide sequence of the coding region designatedzmo1432 in the published Zymomonas mobilis genomic sequence (NCBIReference: NC_(—)006526.2).

SEQ ID NO:2 is the amino acid sequence encoded by the coding regiondesignated zmo1432 in the published Zymomonas mobilis genomic sequence(NCBI Reference: NC_(—)006526.2).

SEQ ID NO:3 is the amino acid sequence of SEQ ID NO:2 but with the aminoacid at position No. 366 changed to arginine.

SEQ ID NO:4 is the amino acid sequence of SEQ ID NO:2 but with the aminoacid at position No. 117 changed to phenylalanine.

SEQ ID NO:5 is the amino acid sequence of the Burkholderia cenocepaciaprotein encoded by fusC (Accession P24128).

SEQ ID NO:6 is the amino acid sequence of the Klebsiella oxytoca fusaricacid detoxification protein (Accession: Q48403)

SEQ ID NO:7 is the amino acid sequence of the Burkholderia cenocepaciaprotein encoded by Bcenmc03_(—)1426. SEQ ID NO:8 is the amino acidsequence of the E. coli protein AaeB.

SEQ ID NO:9 is the amino acid sequence of the immature Xyn3 whichincorporates a predicted signal sequence corresponding to positions 1 to16.

SEQ ID NO:10 is the amino acid sequence of the immature Fv3A whichincorporates a predicted signal sequence corresponding to positions 1 to23.

SEQ ID NO: 11 is the amino acid sequence of the immature Fv43D, whichincorporates a predicted signal sequence corresponding to positions 1 to20.

SEQ ID NO:12 is the amino acid sequence of the immature Fv51A whichincorporates a predicted signal sequence corresponding to positions 1 to19.

DETAILED DESCRIPTION

The present invention describes adaptation of xylose-utilizing Zymomonascells in biomass hydrolysate medium, to improve growth and ethanolproduction. Characterization of mutations in adapted strains identifiedmutations characteristic for the improvement, which may be engineered innon-adapted xylose-utilizing Zymomonas strains. These strains are usedto make ethanol more efficiently, to produce ethanol as a fossil fuelreplacement.

The following abbreviations and definitions will be used for theinterpretation of the specification and the claims.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, are intended to cover a non-exclusive inclusion. Forexample, a composition, a mixture, process, method, article, orapparatus that comprises a list of elements is not necessarily limitedto only those elements but may include other elements not expresslylisted or inherent to such composition, mixture, process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive or and not to an exclusive or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the indefinite articles “a” and “an” preceding an element orcomponent of the invention are intended to be nonrestrictive regardingthe number of instances (i.e. occurrences) of the element or component.Therefore “a” or “an” should be read to include one or at least one, andthe singular word form of the element or component also includes theplural unless the number is obviously meant to be singular.

As used herein “xylose-utilizing Zymomonas cell(s)” refers to a cell orcells of a strain that are genetically engineered to express enzymesconferring the ability to use xylose as a carbohydrate source forfermentation.

The term “adapted strain” refers to a microorganism that has beenselected for growth under particular conditions in order to improve it'sability to grow and produce a product in those conditions.

As used herein “corresponding non-adapted strain” refers to the originalxylose-utilizing Zymomonas strain that is a strain from which improvedstrains are produced using the biomass hydrolysate adaptation processdisclosed herein.

As used herein “feeding growth medium” refers to the medium that isadded into the continuous culture vessel.

The term “lignocellulosic biomass” or “biomass” refers to anylignocellulosic material and includes materials comprising cellulose,hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides.Biomass may also comprise additional components, such as protein and/orlipid. Biomass may be derived from a single source, or biomass cancomprise a mixture derived from more than one source; for example,biomass could comprise a mixture of corn cobs and corn stover, or amixture of grass and leaves. Lignocellulosic biomass includes, but isnot limited to, bioenergy crops, agricultural residues, municipal solidwaste, industrial solid waste, sludge from paper manufacture, yardwaste, wood and forestry waste. Examples of biomass include, but are notlimited to, corn cobs, crop residues such as corn husks, corn stover,grasses, wheat straw, barley straw, hay, rice straw, switchgrass, wastepaper, sugar cane bagasse, sorghum plant material, soybean plantmaterial, components obtained from milling of grains, trees, branches,roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables,fruits, and flowers.

As used herein “biomass hydrolysate” and “cellulosic hydrolysate” referto a product produced from biomass, which is cellulosic material,typically through pretreatment and saccharification processes.Fermentable sugars are present in the hydrolysate, as well as otherproducts.

As used herein “biomass hydrolysate medium” refers to medium whichcontains at least about 50% hydrolysate prepared from cellulosic and/orlignocellulosic biomass. Hydrolysate is prepared by saccharification ofbiomass, typically preceded by pretreatment. In addition to thehydrolysate, the medium may include defined components for biocatalystgrowth and production.

“Xyn3” is a GH10 family xylanase from Trichoderma reesei. Xyn3 (SEQ IDNO:9) was shown to have endoxylanase activity indirectly by observationof its ability to catalyze increased xylose monomer production in thepresence of xylobiosidase when the enzymes act on pretreated biomass oron isolated hemicellulose.

‘Fv3A” is a GH3 family enzyme from Fusarium verticillioides. Fv3A (SEQID NO:10) was shown to have β-xylosidase activity, for example, in anenzymatic assay using p-nitrophenyl-β-xylopyranoside, xylobiose, mixedlinear xylo-oligomers, branched arabinoxylan oligomers fromhemicellulose, or dilute ammonia pretreated corncob as substrates.

“Fv43D” is a GH43 family enzyme from Fusarium verticillioides. Fv43D(SEQ ID NO:11) was shown to have β-xylosidase activity in, for example,an enzymatic assay using p-nitrophenyl-β-xylopyranoside, xylobiose,and/or mixed, linear xylo-oligomers as substrates.

“Fv51A” is a GH51 family enzyme from Fusarium verticillioides. Fv51A(SEQ ID NO:12) was shown to have L-α-arabinofuranosidase activity in,for example, an enzymatic assay using4-nitrophenyl-α-L-arabinofuranoside as a substrate.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein or functional RNA molecule, which may optionally includeregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. “Native gene” or “wildtype gene” refers to a gene as found in nature with its own regulatorysequences. “Chimeric gene” refers to any gene that is not a native gene,comprising regulatory and coding sequences that are not found togetherin nature. Accordingly, a chimeric gene may comprise regulatorysequences and coding sequences that are derived from different sources,or regulatory sequences and coding sequences derived from the samesource, but arranged in a manner different than that found in nature.“Endogenous gene” refers to a native gene in its natural location in thegenome of an organism. A “foreign” gene refers to a gene not normallyfound in the host organism, but that is introduced into the hostorganism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes.

The term “genetic construct” refers to a nucleic acid fragment thatencodes for expression of one or more specific proteins or functionalRNA molecules. In the gene construct the gene may be native, chimeric,or foreign in nature. Typically a genetic construct will comprise a“coding sequence”. A “coding sequence” refers to a DNA sequence thatencodes a specific amino acid sequence.

“Promoter” or “Initiation control regions” refers to a DNA sequencecapable of controlling the expression of a coding sequence or functionalRNA. In general, a coding sequence is located 3′ to a promoter sequence.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic DNA segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmentalconditions. Promoters which cause a gene to be expressed in most celltypes at most times are commonly referred to as “constitutivepromoters”.

The term “expression”, as used herein, refers to the transcription andstable accumulation of coding (mRNA) or functional RNA derived from agene. Expression may also refer to translation of mRNA into apolypeptide. “Overexpression” refers to the production of a gene productin transgenic organisms that exceeds levels of production in normal ornon-transformed organisms.

The term “transformation” as used herein, refers to the transfer of anucleic acid fragment into a host organism, resulting in geneticallystable inheritance. The transferred nucleic acid may be in the form of aplasmid maintained in the host cell, or some transferred nucleic acidmay be integrated into the genome of the host cell. Host organismscontaining the transformed nucleic acid fragments are referred to as“transgenic” or “recombinant” or “transformed” organisms.

The terms “plasmid” and “vector” as used herein, refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “selectable marker” means an identifying factor, usually anantibiotic or chemical resistance gene, that is able to be selected forbased upon the marker gene's effect, i.e., resistance to an antibiotic,wherein the effect is used to track the inheritance of a nucleic acid ofinterest and/or to identify a cell or organism that has inherited thenucleic acid of interest.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: 1.) Computational MolecularBiology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.)Biocomputing: Informatics and Genome Protects (Smith, D. W., Ed.)Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: NY (1991).

Preferred methods to determine identity are designed to give the bestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the MegAlign™ program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequencesis performed using the “Clustal method of alignment” which encompassesseveral varieties of the algorithm including the “Clustal V method ofalignment” corresponding to the alignment method labeled Clustal V(described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in theMegAlign™ program of the LASERGENE bioinformatics computing suite(DNASTAR Inc.). For multiple alignments, the default values correspondto GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters forpairwise alignments and calculation of percent identity of proteinsequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2,GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of thesequences using the Clustal V program, it is possible to obtain a“percent identity” by viewing the “sequence distances” table in the sameprogram. Additionally the “Clustal W method of alignment” is availableand corresponds to the alignment method labeled Clustal W (described byHiggins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al.,Comput. Appl. Biosci. 8:189-191 (1992)) and found in the MegAlign™ v6.1program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.).Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTHPENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5,Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). Afteralignment of the sequences using the Clustal W program, it is possibleto obtain a “percent identity” by viewing the “sequence distances” tablein the same program.

Producing Zymomonas Strains with Improved Fermentation in HydrolysateMedium

The invention provides a recombinant, xylose-utilizing,ethanol-producing microorganism of the genus Zymomonas, having at leastone genetic modification in the zmo1432 open reading frame. The effectof this mutation is to express a polypeptide that improves the behaviorof the strain in a hydrolysate medium, increasing the strain's toleranceto various growth inhibitors in the hydrolysate and increasing the yieldof ethanol. The improvements in fermentation behavior have been linkedto mutations in the zmo 1432 region of the Zymomonas genome, (NCBIReference: NC_(—)006526.2), defined herein as SEQ ID NO:1, encoding thepolypeptide of SEQ ID NO:2.

Accordingly it is put forth here that improved fermentation inhydrolysate medium may be conferred to Zymomonas strains that are ableto utilize xylose as a carbon source and that produce ethanol byintroducing at least one genetic modification in an open reading frame(ORF) that encodes a protein having at least about 95% amino acididentity to SEQ ID NO:2, prior to modification. The protein may have atleast about 95%, 96%, 96%, 98%, 99%, or 100% identity to SEQ ID NO:2.Any genetic modification in said protein may be made which increasesethanol production by the strain harboring said mutant protein in thepresence of hydrolysate medium. Increase in ethanol production isdetermined by comparison to production by a Zymomonas strain lacking thegenetic modification, under the same conditions of fermentation. Astrain with a genetic modification in said ORF may be readily assayed byone of skill in the art to assess increased ethanol production in thepresence of biomass hydrolysate by methods such as described in Example3 herein. Said improved strain has higher tolerance to biomasshydrolysate, and inhibitors present in biomass hydrolysate, wheretolerance refers to the ability of the strain to grow and produceethanol similarly in media with a specified level of hydrolysate (levelof tolerance) as compared to in media with less or no hydrolysate.Higher tolerance is determined by comparison with a Zymomonas strainlacking the genetic modification.

In one embodiment the genetic modification results in an alteration atposition 366 of the amino acid sequence of SEQ ID NO:2 that substitutesarginine for threonine. In another embodiment the genetic modificationresults in an alteration at position 117 of the amino acid sequence ofSEQ ID NO:2 that substitutes phenylalanine for serine. Any change may bemade in the nucleotide sequence which results in the change of codon 366to encode arginine, or results in the change of codon 117 to encodephenylalanine. Codons encoding arginine are CGT, CGC, CGA, CGG, AGA, andAGG. Codons encoding phenylalanine are TTT and TTC.

In one embodiment the genetic modification is in a coding region thathas at least 95% sequence identity to the ORF zmo1432 as named in thepublished Zymomonas mobilis genomic sequence (NCBI Reference:NC_(—)006526.2) which is the complement of nucleotides 1446603 to1448633 having SEQ ID NO:1. The ORF of SEQ ID NO:1 may be calledalternative names, but in any case it may be identified as zmo1432 bycomparison to the sequence of SEQ ID NO:1. There may be some variationin the sequence of coding regions identified as zmo1432 among Zymomonasspecies or strains. Thus a coding region identified as zmo1432 may haveat least about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NO:1 and the coding region with the genetic modification may have atleast about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO:1 prior to genetic modification. This coding region, prior tomodification, is the target for genetic modification.

At least one of the described genetic modifications may be created in aZymomonas strain that is able to utilize xylose as a carbon source andthat produces ethanol by any method known to one of skill in the art.Such methods include by adaptation as described below and in Examples 1and 2 herein, by chemical mutagenesis and screening, and by geneticengineering.

Genetic engineering to introduce a genetic modification in the targetcoding region may be by methods including using double-crossoverhomologous recombination to replace the endogenous target coding regionwith the same coding region harboring a mutation that is describedabove. In homologous recombination, DNA sequences flanking the targetintegration site are placed bounding a spectinomycin-resistance gene orother selectable marker, and the replacement mutant sequence leading toinsertion of the selectable marker and the replacement mutant sequenceinto the target genomic site. The selectable marker is outside of thecoding region so that in the product, the coding region is expressed. Inaddition, the selectable marker may be bounded by site-specificrecombination sites, so that after expression of the correspondingsite-specific recombinase, the resistance gene is excised from thegenome. Particularly suitable for integration of a replacement mutantsequence is transposition using EPICENTRE®'s EZ::Tn in vitrotransposition system, which is used in Examples 1 and 6 of United StatesPatent Publication US-2009-0246846-A1.

Alternatively, expression of the target endogenous coding region may bedisrupted in a cell by a manipulation such as insertion, mutation, ordeletion and a gene expressing the coding region harboring a mutation,as described above, may be introduced into the cell. An example ofdouble-crossover, homologous recombination in Zymomonas used toinactivate a coding region is described in U.S. Pat. No. 7,741,119. Theintroduced gene may be the endogenous gene (with mutant coding region)including its native promoter, or the introduced gene may be a chimericgene comprising an operably linked promoter and a coding region with atleast about 95%, 96%, 97%, 98%, or 99% identity to the disrupted codingregion. Typically a 3′ termination region is included in a chimericgene. Promoters that may be used in Zymomonas include ZmPgap and thepromoter of the Zymomonas enolase gene.

The introduced gene may be maintained on a plasmid, or integrated intothe genome using, for example, homologous recombination, site-directedintegration, or random integration. A gene to be introduced is typicallyconstructed in or transferred to a vector for further manipulations.Vectors are well known in the art. Particularly useful for expression inZymomonas are vectors that can replicate in both E. coli and Zymomonas,such as pZB188 which is described in U.S. Pat. No. 5,514,583. Vectorsmay include plasmids for autonomous replication in a cell, and plasmidsfor carrying constructs to be integrated into bacterial genomes.Plasmids for DNA integration may include transposons, regions of nucleicacid sequence homologous to the target bacterial genome, or othersequences supporting integration. An additional type of vector may be atransposome produced using, for example, a system that is commerciallyavailable from EPICENTRE®. It is well known how to choose an appropriatevector for the desired target host and the desired function.

Host Strain

Any strain of Zymomonas that is able to utilize xylose as a carbonsource and that produces ethanol may be a starting strain for thepresent invention. Such strains are used for adaptation in hydrolysatemedium, for chemical mutagenesis and screening, or are geneticallyengineered to produce the improved strains disclosed herein. Strains ofZymomonas, such as Z. mobilis, that have been engineered to express axylose to ethanol fermentation pathway are particularly useful.Endogenous genes may provide part of the metabolic pathway, or may bealtered by any known genetic manipulation technique to provide a proteinwith enzyme activity useful for xylose metabolism. For example, theendogenous transketolase may complement other introduced enzymeactivities in creating a xylose utilization pathway. Typically fourgenes may be introduced into a Zymomonas strain, such as Z. mobilis, forexpression of four enzymes involved in xylose metabolism as described inU.S. Pat. No. 5,514,583, which is herein incorporated by reference.These include genes encoding xylose isomerase, which catalyzes theconversion of xylose to xylulose and xylulokinase, which phosphorylatesxylulose to form xylulose 5-phosphate. In addition, transketolase andtransaldolase, two enzymes of the pentose phosphate pathway, convertxylulose 5-phosphate to intermediates that couple pentose metabolism tothe glycolytic Entner-Douderoff pathway permitting the metabolism ofxylose to ethanol. DNA sequences encoding these enzymes may be obtainedfrom any of numerous microorganisms that are able to metabolize xylose,such as enteric bacteria, and some yeasts and fungi. Sources for thecoding regions include Xanthomonas, Klebsiella, Escherichia,Rhodobacter, Flavobacterium, Acetobacter, Gluconobacter, Rhizobium,Agrobacterium, Salmonella, Pseudomonads, and Zymomonas. Particularlyuseful are the coding regions of E. coli.

The encoding DNA sequences are operably linked to promoters that areexpressed in Z. mobilis cells such as the promoters of Z. mobilisglyceraldehyde-3-phosphate dehydrogenase (GAP promoter), and Z. mobilisenolase (ENO promoter). The coding regions may individually be expressedfrom promoters, or two or more coding regions may be joined in an operonwith expression from the same promoter. The resulting chimeric genes maybe introduced into Zymomonas and maintained on a plasmid, or integratedinto the genome using, for example, homologous recombination,site-directed integration, or random integration. Xylose-utilizingstrains that are of particular use include ZM4(pZB5) (described in U.S.Pat. No. 5,514,583, U.S. Pat. No. 6,566,107, and U.S. Pat. No.5,571,2133, and incorporated by reference herein), 8b (United StatesPatent Application U.S. 2003/0162271; Mohagheghi et al., (2004)Biotechnol. Lett. 25; 321-325), as well as ZW658 (ATCC PTA-7858), ZW800,ZW801-4, ZW801-5, and ZW801-6 (described in commonly owned andco-pending United States Patent Application Publication U.S.2008-0286870 A1, which is herein incorporated by reference).

Zymomonas strains that are additionally engineered to utilize othersugars that are not natural substrates, may also be used in the presentprocess. An example is a strain of Z. mobilis engineered for arabinoseutilization as described in U.S. Pat. No. 5,843,760, which is hereinincorporated by reference.

Adaptation

For adaptation, a xylose-utilizing strain of Zymomonas (a startingstrain as described above) is continuously grown in a medium containingbiomass hydrolysate. Biomass hydrolysate is produced by saccharificationof biomass. Typically the biomass is pretreated prior tosaccharification. Biomass may be treated by any method known by oneskilled in the art to produce fermentable sugars in a hydrolysate.Typically the biomass is pretreated using physical and/or chemicaltreatments, and saccharified enzymatically. Physical and chemicaltreatments may include grinding, milling, cutting, base treatment suchas with ammonia or NaOH, and acid treatment. Particularly useful is alow ammonia pretreatment where biomass is contacted with an aqueoussolution comprising ammonia to form a biomass-aqueous ammonia mixturewhere the ammonia concentration is sufficient to maintain an alkaline pHof the biomass-aqueous ammonia mixture but is less than about 12 wt. %relative to dry weight of biomass, and where dry weight of biomass is atleast about 15 wt % solids relative to the weight of the biomass-aqueousammonia mixture, as disclosed in co-pending and commonly owned UnitedStates Patent Application Publication US-20070031918-A1, which is hereinincorporated by reference.

Enzymatic saccharification typically makes use of an enzyme compositionor blend to break down cellulose and/or hemicellulose and to produce ahydrolysate containing sugars such as, for example, glucose, xylose, andarabinose. Saccharification enzymes are reviewed in Lynd, L. R., et al.(Microbiol. Mol. Biol. Rev., 66:506-577, 2002). At least one enzyme isused, and typically a saccharification enzyme blend is used thatincludes one or more glycosidases. Glycosidases hydrolyze the etherlinkages of di-, oligo-, and polysaccharides and are found in the enzymeclassification EC 3.2.1.x (Enzyme Nomenclature 1992, Academic Press, SanDiego, Calif. with Supplement 1 (1993), Supplement 2 (1994), Supplement3 (1995, Supplement 4 (1997) and Supplement 5 [in Eur. J. Biochem.,223:1-5, 1994; Eur. J. Biochem., 232:1-6, 1995; Eur. J. Biochem.,237:1-5, 1996; Eur. J. Biochem., 250:1-6, 1997; and Eur. J. Biochem.,264:610-650 1999, respectively]) of the general group “hydrolases” (EC3). Glycosidases useful in the present method can be categorized by thebiomass components they hydrolyze. Glycosidases useful for the presentmethod include cellulose-hydrolyzing glycosidases (for example,cellulases, endoglucanases, exoglucanases, cellobiohydrolases,β-glucosidases), hemicellulose-hydrolyzing glycosidases (for example,xylanases, endoxylanases, exoxylanases, β-xylosidases,arabino-xylanases, mannases, galactases, pectinases, glucuronidases),and starch-hydrolyzing glycosidases (for example, amylases, α-amylases,β-amylases, glucoamylases, α-glucosidases, isoamylases). In addition, itmay be useful to add other activities to the saccharification enzymeconsortium such as peptidases (EC 3.4.x.y), lipases (EC 3.1.1.x and3.1.4.x), ligninases (EC 1.11.1.x), or feruloyl esterases (EC 3.1.1.73)to promote the release of polysaccharides from other components of thebiomass. It is known in the art that microorganisms that producepolysaccharide-hydrolyzing enzymes often exhibit an activity, such as acapacity to degrade cellulose, which is catalyzed by several enzymes ora group of enzymes having different substrate specificities. Thus, a“cellulase” from a microorganism may comprise a group of enzymes, one ormore or all of which may contribute to the cellulose-degrading activity.Commercial or non-commercial enzyme preparations, such as cellulase, maycomprise numerous enzymes depending on the purification scheme utilizedto obtain the enzyme.

Saccharification enzymes may be obtained commercially. Such enzymesinclude, for example, Spezyme® CP cellulase, Multifect® xylanase,Accelerase® 1500, and Accellerase® DUET (Danisco U.S. Inc., GenencorInternational, Rochester, N.Y.). In addition, saccharification enzymesmay be unpurified and provided as a cell extract or a whole cellpreparation. The enzymes may be produced using recombinantmicroorganisms that have been engineered to express one or moresaccharifying enzymes.

Additional enzymes for saccharification include, for example, glycosylhydrolases such as members of families GH3, GH39, GH43, GH55, GH10, andGH11. GHs are a group of enzymes that hydrolyze the glycosidic bondbetween two or more carbohydrates, or between a carbohydrate and anoncarbohydrate moiety. Families of GHs have been classified based onsequence similarity and the classification is available in theCarbohydrate-Active enzyme (CAZy) database (Cantarel et al. (2009)Nucleic Acids Res. 37 (Database issue):D233-238). Certain of theseenzymes are able to act on various substrates and have demonstratedeffecacy as saccharification enzymes. Glycoside hydrolase family 3(“GH3”) enzymes have a number of known activities, including, forexample, β-glucosidase (EC:3.2.1.21); β-xylosidase (EC:3.2.1.37);N-acetyl β-glucosaminidase (EC:3.2.1.52); glucan β-1,3-glucosidase(EC:3.2.1.58); cellodextrinase (EC:3.2.1.74); exo-1,3-1,4-glucanase(EC:3.2.1); and/or β-galactosidase (EC 3.2.1.23) activities. Glycosidehydrolase family 39 (“GH39”) enzymes also have a number of knownactivities, including, for example, α-L-iduronidase (EC:3.2.1.76) and/orβ-xylosidase (EC:3.2.1.37) activities. Glycoside hydrolase family 43(“GH43”) enzymes have a number of known activities including, forexample, L-α-arabinofuranosidase (EC 3.2.1.55); β-xylosidase (EC3.2.1.37); endoarabinanase (EC 3.2.1.99); and/or galactan1,3-β-galactosidase (EC 3.2.1.145) activities. Glycoside hydrolasefamily 51 (“GH51”) enzymes are known to have, for example,L-α-arabinofuranosidase (EC 3.2.1.55) and/or endoglucanase (EC 3.2.1.4)activities. Glycoside hydrolase family 10 (“GH10”) have been describedin detail in Schmidt et al., 1999, Biochemistry 38:2403-2412 and LoLeggio et al., 2001, FEBS Lett 509: 303-308) and the Glycoside hydrolasefamily 11 (“GH11”) have been described in Hakouvainen et al., 1996,Biochemistry 35:9617-24.

In the present adaptation process, xylose-utilizing Zymomonas iscontinuously grown in the presence of increasing proportions of biomasshydrolysate in the growth medium as described in Examples 1 and 2herein. At periodic intervals samples are taken and assayed forperformance in hydrolysate medium, including for xylose and glucoseutilization, and for ethanol production. Multiple rounds of adaptationin a medium containing increasing proportions of hydrolysates, as wellas other stress components such as acetate and ethanol, may be used toproduce strains with improved performance, such as those describedabove.

Fermentation of Improved Xylose-Utilizing Strain

An engineered xylose-utilizing and ethanol producing Zymomonas strainwith at least one genetic modification described herein may be used infermentation to produce ethanol. The Z. mobilis strain is brought incontact with medium that contains biomass hydrolysate that includessugars comprising glucose and xylose. At least a portion of the sugarsare derived from pretreated and saccharified cellulosic orlignocellulosic biomass. Additional sugars and/or other media componentsmay be included in the medium.

When the sugars concentration is high such that growth is inhibited, themedium includes sorbitol, mannitol, or a mixture thereof as disclosed incommonly owned U.S. Pat. No. 7,629,156. Galactitol or ribitol mayreplace or be combined with sorbitol or mannitol. The Z. mobilis growsin the medium where fermentation occurs and ethanol is produced. Thefermentation is run without supplemented air, oxygen, or other gases(which may include conditions such as anaerobic, microaerobic, ormicroaerophilic fermentation), for at least about 24 hours, and may berun for 30 or more hours. The timing to reach maximal ethanol productionis variable, depending on the fermentation conditions. The fermentationsmay be run at temperatures that are between about 30° C. and about 37°C., at a pH of about 4.5 to about 7.5.

The present Z. mobilis may be grown in medium containing mixed sugarsincluding xylose in laboratory scale fermenters, and in scaled upfermentation where commercial quantities of ethanol are produced. Wherecommercial production of ethanol is desired, a variety of culturemethodologies may be applied. For example, large-scale production fromthe present Z. mobilis strains may be produced by both batch andcontinuous culture methodologies. A classical batch culturing method isa closed system where the composition of the medium is set at thebeginning of the culture and not subjected to artificial alterationsduring the culturing process. Thus, at the beginning of the culturingprocess the medium is inoculated with the desired organism and growth ormetabolic activity is permitted to occur adding nothing to the system.Typically, however, a “batch” culture is batch with respect to theaddition of carbon source and attempts are often made at controllingfactors such as pH and oxygen concentration. In batch systems themetabolite and biomass compositions of the system change constantly upto the time the culture is terminated. Within batch cultures cellsmoderate through a static lag phase to a high growth log phase andfinally to a stationary phase where growth rate is diminished or halted.If untreated, cells in the stationary phase will eventually die. Cellsin log phase are often responsible for the bulk of production ofethanol.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch culture processes are also suitable for growth of the presentZ. mobilis strains and comprise a typical batch system with theexception that the substrate is added in increments as the cultureprogresses. Measurement of the actual substrate concentration inFed-Batch systems is difficult and is therefore estimated on the basisof the changes of measurable factors such as pH and the partial pressureof waste gases such as CO₂. Batch and Fed-Batch culturing methods arecommon and well known in the art and examples may be found inBiotechnology: A Textbook of Industrial Microbiology, Crueger, Crueger,and Brock, Second Edition (1989) Sinauer Associates, Inc., Sunderland,Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36, 227,(1992), herein incorporated by reference.

Commercial production of ethanol may also be accomplished with acontinuous culture. Continuous cultures are open systems where culturemedium is added continuously to a bioreactor and an equal amount ofconditioned medium is removed simultaneously for processing. Continuouscultures generally maintain the cells at a constant high liquid phasedensity where cells are primarily in log phase growth. Alternatively,continuous culture may be practiced with immobilized cells where carbonand nutrients are continuously added, and valuable products, by-productsor waste products are continuously removed from the cell mass. Cellimmobilization may be performed using a wide range of solid supportscomposed of natural and/or synthetic materials as is known to oneskilled in the art.

Particularly suitable for ethanol production is a fermentation regime asfollows. The desired Z. mobilis strain of the present invention is grownin shake flasks in semi-complex medium at about 30° C. to about 37° C.with shaking at about 150 rpm in orbital shakers and then transferred toa 10 L seed fermentor containing similar medium. The seed culture isgrown in the seed fermentor anaerobically until OD₆₀₀ is between 3 and10, when it is transferred to the production fermentor where thefermentation parameters are optimized for ethanol production. Typicalinoculum volumes transferred from the seed tank to the production tankrange from about 2% to about 20% v/v. Fermentation medium for thepresent strains contains biomass hydrolysate in at least about 50% andmay be supplemented with other nutrients, as known to one of skill inthe art. A final concentration of about 5 mM sorbitol or mannitol ispresent in the medium.

The fermentation is controlled at pH 5.0-6.0 using caustic solution(such as ammonium hydroxide, potassium hydroxide, or sodium hydroxide)and either sulfuric or phosphoric acid. The temperature of the fermentoris controlled at 30° C.-35° C. In order to minimize foaming, antifoamagents (any class—silicone based, organic based etc) are added to thevessel as needed. An antibiotic, for which there is an antibioticresistant marker in the strain or to which the strain is resistant, suchas kanamycin, may be used optionally to minimize contamination.

Any set of conditions described above, and additionally variations inthese conditions that are well known in the art, are suitable conditionsfor production of ethanol by a xylose-utilizing recombinant Zymomonasstrain.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is disclosed herein that xylose-utilizing Zymomonas strains withimproved utilization of glucose and xylose, and production of ethanol,in the presence of biomass hydrolysate can be obtained by a process ofadaptation and screening in biomass hydrolysate medium. Increase inglucose and xylose utilization, and ethanol production, is measured bycomparison to glucose and xylose utilization by the xylose-utilizingcorresponding non-adapted strain. The corresponding non-adapted strainis the strain used as the starting strain for the hydrolysate adaptationprocess.

Zymomonas mobilis strains were isolated from hydrolysate adaptationexperiments as described in Examples 1 and 2 herein. Two isolatedstrains were named Adapted 5-6 (or AR25-6) and Adapted 7-31 (orAR37-31), and were further characterized. When grown in corn cobhydrolysate as described in Example 3 herein, these strains utilizedglucose and xylose more rapidly than the corresponding non-adaptedstrain ZW705. At 21 hours of a fermentation run, about 20% more glucosehad been utilized. In addition, at 21 hours about 22% more ethanol wasproduced, while at 52 hours about 21% more xylose had been used and 5%more ethanol was produced. In general, the exact percent increases inglucose utilization, xylose utilization, and ethanol production in astrain with new genetic changes of the Adapted 5-6 or Adapted 7-31strains, that are described below, will depend on many factors. Thesefactors include the conditions of the fermentation as well as thegenetic characteristics of the strain that are in addition to the newgenetic changes disclosed herein.

To identify any changes in the genomes of the Adapted 5-6 and Adapted7-31 strains, the genomes were sequenced. By comparison of these genomicsequences to genomic sequences of the corresponding starting strain,ZW705 (described in United States Patent Publication US20110014670A1),wild type strain ZW1 (ATCC 31821), and the published Zymomonas mobilissequence of strain ATCC 31821 (Seo et. al, Nat. Biotech. 23:63-8 2005;NCBI Reference: NC_(—)006526.2), it was determined that both strains hada single, new mutation in the same coding region. This coding region isidentified as the zmo1432 open reading frame (ORF) in the publishedZymomonas mobilis genomic sequence (NCBI Reference: NC_(—)006526.2) andis the complement of nucleotides 1446603 to 1448633 having SEQ ID NO:1.

Adapted 5-6 has a mutation at position No. 1097 in SEQ ID NO:1 that is achange from C to G. This results in the change of codon 366 from ACAencoding threonine to AGA encoding arginine in the zmo1432 encodedprotein (SEQ ID NO:2) resulting in the protein of SEQ ID NO:3 wherearginine is substituted for threonine No. 366. Adapted 7-31 has amutation at position No. 350 in SEQ ID NO:1 that is a change from C toT. This results in the change of codon 117 from TCT encoding serine toTTT encoding phenylalanine in the zmo1432 encoded protein (SEQ ID NO:2))resulting in the protein of SEQ ID NO:4 where phenylalanine issubstituted for serine No. 117.

The hypothetical coding region zmo1432 (or ORF) is annotated in theZymomonas mobilis complete genome sequence (NCBI Reference:NC_(—)006526.2) as encoding a “fusaric acid resistance protein”. It isannotated as having a fusaric acid resistance protein conserved regionwhich is protein motif: PFAM:PF04632 (Wellcome Trust Sanger Institute,Genome Research Limited, Hinxton, England). This motif is found inproteins associated with fusaric acid resistance from Burkholderiacepacia (Swiss-Prot::P24128 (SEQ ID NO:5); Utsumi et al. (1991) Agric.Biol. Chem. 55:1913-1918; the organism was renamed from Pseudomonascepacia) and Klebsiella oxytoca (Swiss-Prot::Q48403 (SEQ ID NO:6);Toyoda et al. (1991) J Phytopathol. 133:165-277), which are likely to bemembrane transporter proteins.

A region named Bcenm03_(—)1426 of the Burkholderia cepacia chromosome 1complete sequence is annotated as encoding a putative fusaric acidresistance transporter protein (Accession YP_(—)001764723; Copeland etal. submitted Feb. 27, 2008). This protein (SEQ ID NO:7) has similarityto the zmo1432 encoded protein as described in Example 4 herein. Inaddition, the E. coli protein AaeB (SEQ ID NO:8), which is a componentof an aromatic carboxylic acid efflux pump (VanDyk et al J. Bact.186:7196-7204 (2004)), has similarity to the zmo1432 encoded protein, asdescribed in Example 4 herein. Thus a transporter protein of Zymomonasmay be the target of mutations that improve glucose and xyloseutilization, and ethanol production in xylose-utilizing Zymomonasstrains that are grown in hydrolysate medium.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

The meaning of abbreviations is as follows: “kb” means kilobase(s), “bp”means base pairs, “nt” means nucleotide(s), “hr” means hour(s), “min”means minute(s), “sec” means second(s), “d” means day(s), “L” meansliter(s), “ml” means milliliter(s), “μL” or “μl” means microliter(s),“μg” means microgram(s), “ng” means nanogram(s), “g” means gram(s), “mM”means millimolar, “μM” means micromolar, “nm” means nanometer(s), “mmol”means micromole(s), “pmol” means picomole(s), “OD600” is optical densityat 600 nm.

General Methods

Turbidostat

Adaptation was in a turbidostate (U.S. Pat. No. 6,686,194; Heurisko USA,Inc. Newark, Del.), which is a continuous flow culture device where theconcentration of cells in the culture was kept constant by controllingthe flow of medium into the culture, such that the turbidity of theculture was kept within specified narrow limits. Two media wereavailable to the growing culture in the continuous culture device, aresting medium (Medium A) and a challenge medium (Medium B). A culturewas grown on a resting medium in a growth chamber to a turbidity setpoint and then was diluted at a dilution rate set to maintain that celldensity. Dilution was performed by adding media at a defined volume onceevery 10 minutes. When the turbidostat entered a media challenge mode,the choice of adding challenge medium or a resting medium was made basedon the rate of return to the set point after the previous mediaaddition. The steady state concentration of medium in the growth chamberwas a mix of Medium A and Medium B, with the proportions of the twomedia dependent upon the rate of draw from each medium that allowedmaintenance of the set cell density at the set dilution rate. A sampleof cells representative of the population in the growth chamber wasrecovered from the outflow of the turbidostat (in a trap chamber) atweekly intervals. The cell sample was grown once in MRM3G6 medium andsaved as a glycerol stock at −80° C.

Enzymes

Spezyme® CP-100, Multifect® CX12L, and Accellerase® 1500 were fromDanisco U.S. Inc., Genencor (Rochester, N.Y.).

Novozyme 188 was from Novozymes (2880 Bagsvaerd, Denmark).

Additional enzymes used in the saccharification process(es) herein werethe glycosyl hydrolases (GH) Xyn3, Fv3A, Fv51A and Fv43D. Xyn3 (SEQ IDNO:9) is a GH10 family xylanase from Trichoderma reesei, Fv3A (SEQ IDNO:10) is a GH3 family enzyme from Fusarium verticillioides, Fv43D (SEQID NO:11) is a GH43 family enzyme from Fusarium verticillioides, andFv51A (SEQ ID NO:12) is a GH51 family of enzyme from Fusariumverticillioides.

Media.

Corn cob hydrolysate used in the adaptation was prepared first by diluteammonia pretreatment of ground corn cob. A horizontal Littleford Day130L reactor vessel containing a jacket for passing steam around thebody of the vessel and one of the sides (Littleford Day, Inc., Florence,Ky.) was loaded with milled cob. Vacuum was applied to the vessel toreach 0.1 atm prior to introduction of a 29 wt % ammonium hydroxidesolution and water near the top of the vessel to give a 6 wt % NH₃relative to dry weight biomass. Steam was introduced near the top of thevessel to raise the internal vessel temperature to 145° C. Thistemperature was held for 20 mins. At the end of pretreatment, thereactor was depressurized to reach atmospheric pressure. Vacuum(approximately to less than 1 atm) was subsequently applied to lower thetemperature to less than 60° C.

The pretreated cob was then treated with and enzyme consortium allowingfor enzymatic hydrolysis of the cellulose and hemicellulose polymersusing an enzyme mix containing: Spezyme CP-100 at 34 mg protein per g ofglucan in the ptretreated cob; Multifect CX12L at 12.5 mg protein per gof xylan in the pretreated cob; Novozymes 188 at 6.6 mg protein per gglucan in the pretreated cob. The hydrolysis reaction was carried out at25% (weight/volume) pretreated cob dry matter, pH 5.3 and 47° C. Thereaction was stirred continuously and ran for 72 hrs.

Solids were removed from the hydrolysate using an initial, continuouscentrifugation to partially clarify the mixture. The partially clarifiedmix was again centrifuged at 18,000×G for 20 mins then passed firstthrough a 0.45 micron filter followed by a 0.22 micron filter to produceclarified, filter sterilized hydrolysate.

Each of the glucose, xylose and acetate concentrations was determined byHPLC analysis. The clarified hydrolysate had a 68 g/L glucoseconcentration, 46 g/L xylose concentration, and 5 g/L acetateconcentration. The clarified hydrolysate was supplemented with 6.2 g/Lammonium acetate to increase the total ammonium acetate concentrationsuch that the concentration falls within the range of 11 to 12 g/L.Where noted, 0.5% yeast extract (Difco Yeast Extract, Becton, Dickinsonand Co., Sparks, Md.) was added to provide additional nutrients. Thismedium was labeled HYAc/YE. The pH of the HYAc/YE medium was adjusted to5.8 and the medium was filter sterilized.

A hydrolysate medium for 1 L fermentation test was prepared using themethod described above, except that the enzyme composition used in thehydrolysis was changed to an enzyme blend comprising Accellerase® 1500,Xyn3, Fv3A, Fv51A, and Fv43D, which was added to the hydrolysis reactionat 21.3 mg protein/g glucan+xylan. The hydrolysate was used withoutclarification in the 1 L scale fermentations.

Additional Media

MRM3 contains per liter: yeast extract (10 g), KH₂PO₄ (2 g) andMgSO₄.7H₂O (1 g)

MRM3G6.5X4.5NH₄Ac12.3 is MRM3 containing 65 g/L glucose, 45 g/L xylose,12.3 g/L ammonium acetate

G5 or MRM3G5 is MRM3 containing 50 g/L glucose

G10 or MRM3G10 is MRM3 containing 100 g/L glucose

MRM3G2 is MRM3 containing 20 g/L glucose

MRM3X2 is MRM3 containing 20 g/l xylose

halfYEMaxSM:10 g/L Difo yeast extract, 2 g/L KH₂PO₄, 5 g/L MgSO₄.7H₂O,10 mM sorbitol, 150 g/L glucose

For plating studies noted in FIG. 2, MRM3G2 (20 g/L glucose) and MRM3X2(20 g/L xylose) were supplemented with 1.5% agar, heated to dissolve theagar, cooled to 45° C. and poured into petri dishes.

Preparation of Frozen Stock Cultures

Frozen stock cultures were prepared for strain ZW705, weekly turbidostatsample population cultures, and isolated mutant clones. Additionalfrozen stocks were prepared by growing the original frozen stock on G5or G10 medium and using the biomass generated to prepare new frozenstocks.

Inoculation of Seed, Batch Adaptation and Turbidostat Cultures

Frozen stock was used to inoculate overnight G5 or G10 seed cultures.The seed cultures were used to inoculate batch adaptation cultures bycentrifuging the seed culture medium and diluting the cell pellet infresh adaptation medium. For turbidostat inoculation, the entire seedpellet was re-suspended in 10-15 ml of turbidostat resting medium andused to inoculate the turbidostat reactor or 5 ml of overnight seed wasused as a 10% inoculum.

Culture OD600 Measurements

To measure culture OD, samples were diluted in 100 g/L xylose (diluentand blank). The diluted culture was allowed to sit for 15 minutes beforetaking the OD measurement. All OD measurements were at 600 nm.

HPLC Analysis

HPLC analyses were performed with a Waters Alliance HPLC system. Thecolumn used was a Transgenomic ION-300 column (#ICE-99-9850,Transgenomic, Inc) with a BioRad Micro-Guard Cartridge Cation-H(#125-0129, Bio-Rad, Hercules, Calif.). The column was run at 75° C. and0.4 mL/min flow rate using 0.01 NH₂SO₄ as solvent. The concentrations ofstarting sugars and products were determined with a refractive indexdetector using external standard calibration curves.

Strain ZW705 Description

Zymomonas mobilis strainZW705 was produced from strain ZW804-1. ZW801-4is a recombinant xylose-utilizing strain of Z. mobilis that wasdescribed in commonly owned U.S. Pat. No. 7,741,119, which isincorporated herein by reference. Strain ZW801-4 was derived from strainZW800, which was derived from strain ZW658, all as described in U.S.Pat. No. 7,741,119. ZW658 was constructed by integrating two operons,P_(gap)xylAB and P_(gap)taltkt, containing four xylose-utilizing genesencoding xylose isomerase, xylulokinase, transaldolase andtransketolase, into the genome of ZW1 (ATCC 31821) via sequentialtransposition events, and followed by adaptation on selective mediacontaining xylose (U.S. Pat. No. 7,629,156). ZW658 was deposited as ATCCPTA-7858. In ZW658, the gene encoding glucose-fructose oxidoreductasewas insertionally-inactivated using host-mediated, double-crossover,homologous recombination and spectinomycin resistance as a selectablemarker to create ZW800 (U.S. Pat. No. 7,741,119). The spectinomycinresistance marker, which was bounded by loxP sites, was removed by sitespecific recombination using Cre recombinase to create ZW801-4.

Cultures of Z. mobilis strain ZW801-4 were adapted for growth understress conditions of medium containing ammonium acetate to produce ZW705as described in commonly owned United States Patent PublicationUS20110014670A1, which is incorporated herein by reference. A continuousculture of ZW801-4 was run in 250 ml stirred, pH and temperaturecontrolled fermentors (Sixfors; Bottmingen, Switzerland). The basalmedium for fermentation was 5 g/L yeast extract, 15 mM ammoniumphosphate, 1 g/L magnesium sulfate, 10 mM sorbitol, 50 g/L xylose and 50g/L glucose. Adaptation to growth in the presence of high concentrationsof acetate and ammonia was effected by gradually increasing theconcentration of ammonium acetate added to the above continuous culturemedia while maintaining an established growth rate as measured by thespecific dilution rate over a period of 97 days. Ammonium acetate wasincreased to a concentration of 160 mM. Further increases in ammoniumion concentration were achieved by addition of ammonium phosphate to afinal total ammonium ion concentration of 210 mM by the end of 139 daysof continuous culture. Strain ZW705 was isolated from the adaptedpopulation by plating to single colonies and amplification of one chosencolony.

Example 1 Adaptation to Corn Cob Hydrolysate Using an Automated, CellDensity Controlled Continuous Culture Apparatus

The turbidostat described in General Methods was used to adapt Zymomonasmobilis cultures to growth in corn cob hydrolysate medium. A culture ofstrain ZW705 was grown in the turbidostat described in General Methodsto an arbitrary turbidity set point that dictated that the culture useall of the glucose and approximately half of the xylose present in theincoming media to meet the set point cell density at the set dilutionrate. Using resting medium that was 50% HYAc/YE and 50%MRM3G6.5X4.5NH₄Ac12.3. The turbidostat was run as described in GeneralMethods using as challenge medium HYAc/YE. Cell samples were takenweekly for 6 weeks from the trap chamber.

After 6 weeks of continuous culture, samples from the weekly saved cellstocks were revived in MRM3G6 and grown to about 1 OD600 in 10 ml staticcultures at 33° C. These were used to inoculate 12 ml cultures ofHYAc/YE medium to approximately 0.4 OD600 nm, and the cultures weregrown at 30° C. Samples were taken at different times as in Table 1, andassayed for OD600, sugar consumption, and ethanol production. Theresults are shown in Table 1.

TABLE 1 Analysis of weekly trubidostat samples grown in 12 ml HYAc/YEfermentations remaining remaining Sampling OD glucose xylose ethanolculture time 600 nm (g/L) (g/L) (g/L) ZW705 0 0.400 69.8 46.0 0.0 240.760 52.8 45.4 8.4 48 2.140 5.4 30.7 38.2 72 2.300 2.3 16.0 45.1 week 10 0.417 69.8 46.0 0.0 24 0.78 48.8 45.6 9.7 48 1.430 4.8 34.2 35.6 721.890 2.4 25.7 37.7 week 2 0 0.351 69.8 46.0 0.0 24 0.810 53.2 46.1 7.748 1.670 7.6 36.1 33.5 72 1.710 2.7 26.6 39.3 week 3 0 0.395 69.8 46.00.0 24 1.090 42.9 44.3 13.4 48 1.920 2.9 27.3 40.2 72 2.410 1.9 19.742.1 week 4 0 0.386 69.8 46.0 0.0 24 1.070 42.2 44.2 13.7 48 1.510 3.330.7 38.4 72 2.010 2.1 25.2 39.4 week 5 0 0.328 69.8 46.0 0.0 24 0.92052.7 45.5 7.7 48 1.350 8.5 36.2 34.1 72 1.650 2.9 25.8 40.4 week 6 00.402 69.8 46.0 0.0 24 0.600 55.5 45.9 6.9 48 1.360 13.9 39.8 29.1 721.530 4.1 31.6 37.5

All cultures retained the ability to grow on xylose but the rate ofxylose use seemed to have decreased after the 3^(rd) week of adaptation.Single colonies were isolated from the culture of cells sampled at theend of week 3. Colonies were isolated by growing the retained glycerolstock in MRM3G5, then plating on MRM3X2 plates. Single colonies werereplica patched onto MRM3X2 and MRM3G2 plates. Large, dense patches onboth carbon sources were chosen as strains and maintained as frozenglycerol stocks. Selected strains were grown in 12 ml test cultures asdescribed for the frozen stock population test described above. Resultsfor six strains and ZW705 are shown in Table 2.

TABLE 2 Analysis of isolated pure cultures from week 3 of the turbidosatadaptation of ZW705 grown in 12 ml HYAc/YE fermentations remainingremaining Sampling OD glucose xylose ethanol Strain time 600 nm (g/L)(g/L) (g/L) ZW705 0 0.383 74.9 44.4 0.0 24 0.450 51.1 43.4 13.4 48 1.3505.7 35.1 31.4 72 2.770 1.9 17.1 49.0 12-18X- 0 0.333 74.9 44.4 0.0 1-1024 0.760 47.7 44.4 14.6 48 2.530 3.1 28.3 36.7 72 2.800 1.7 12.1 50.412-18X- 0 0.400 74.9 44.4 0.0 2-36 24 0.900 46.9 44.3 15.1 48 2.220 2.824.2 38.9 72 3.430 1.8 9.1 53.3 12-18X- 0 3.820 74.9 44.4 0.0 5-34 240.530 52.7 44.9 13.1 48 2.430 4.3 30.3 35.7 72 3.050 1.9 13.0 51.112-18X- 0 0.401 74.9 44.4 0.0 6-9 24 0.530 51.2 44.4 13.1 48 2.590 3.228.7 36.6 72 3.050 1.8 11.4 52.5 12-18X- 0 0.351 74.9 44.4 0.0 7-43 240.600 49.6 43.9 14.3 48 2.200 3.1 26.9 38.3 72 3.330 1.7 9.9 53.812-18X- 0 0.409 74.9 44.4 0.0 8-19 24 0.749 45.2 44.4 16.0 48 2.230 3.427.3 34.8 72 2.910 1.7 11.7 52.1

All of the selected strains produced more ethanol in the 12 ml test thandid the strain that entered adaptation (ZW705). The strains usedslightly more xylose than the parent strain. Strain 12-18X-2-36 waschosen for an additional round of adaptation.

Example 2 Adaptation to Corn Cob Hydrolysate with Added Ethanol Using anAutomated, Cell Density Controlled Continuous Culture Apparatus

A culture of strain 12-18X-2-36 (described in Example 1) was grown inthe turbidostat as described above for strain ZW705 and in GeneralMethods, except that the resting medium was HYAc/YE and the challengemedium was HYAc/YE+9 weight % ethanol. The turbidostat was run for 4weeks with weekly sampling of the chamber outflow. Frozen cell stockswere made from the outflow samples. Frozen cell stocks were revived inMRM3G6 for testing in 12 ml hydrolysate fermentations under the sameconditions as described for the first turbidosat run, but with startingdensity of about 0.5 OD600. One fermentation was in the same HYAc/YEmedium, and another fermentation was in HYAc/YE to which ethanol wasadded at 30 g per L of medium. Results for both fermentations are shownin Table 3.

TABLE 3 Analysis of weekly trubidostat samples grown in 12 ml HYAc/YE orHYAc/YE + 30 g/L ethanol fermentations HYAc/YE with no added ethanolHYAc/YE with 30 g/L ethanol remaining remaining remaining remainingsampling glucose xylose ethanol OD glucose xylose ethanol culture timeOD 600 nm (g/L) (g/L) (g/L) 600 nm (g/L) (g/L) (g/L) ZW705 0 0.530 75.547.7 0.0 0.530 73.3 48.3 26.2 24 0.890 50.1 48.0 13.7 0.470 63.6 46.827.3 48 2.200 3.9 33.6 43.6 0.760 51.1 45.7 32.7 72 2.500 1.8 19.7 50.31.060 39.0 45.7 39.6 week 1 0 0.513 75.5 47.7 0.0 0.515 73.3 48.3 26.224 1.360 20.1 44.2 30.2 1.370 45.2 46.9 36.3 48 2.200 2.1 27.3 47.71.810 14.7 42.5 50.1 72 2.520 1.7 18.5 50.6 1.920 3.8 40.1 57.7 week 2 00.504 75.5 47.7 0.0 0.504 73.3 48.3 26.2 24 1.140 32.4 47.1 23.2 0.95051.6 46.8 34.1 48 1.910 2.4 27.8 47.7 1.500 19.4 42.8 50.4 72 2.290 1.718.5 51.4 1.780 5.7 38.0 60.5 week 3 0 0.527 75.5 47.7 0.0 0.527 73.348.3 26.2 24 0.640 53.5 48.7 12.3 0.570 62.1 48.2 28.5 48 1.420 7.3 37.739.5 0.600 51.9 46.1 33.2 72 2.010 2.0 25.5 47.3 1.060 35.3 44.8 38.7week 4 0 0.518 75.5 47.7 0.0 0.518 73.3 48.3 26.2 24 0.730 25.5 45.227.8 0.480 64.3 47.7 25.5 48 1.760 2.3 23.7 49.3 0.560 52.8 46.8 32.9 721.890 1.8 15.8 52.6 1.080 38.4 45.2 40.4

All cultures were similar to the ZW705 control in the test fermentationswith no ethanol added to the HYAc/YE medium. In fermentations with 30g/L ethanol added to the HYAc/YE medium, the cultures taken after weeks1 and 2 were much better at utilizing glucose and producing ethanol thanZW705 and the week 3 and 4 samples. The culture stored after week 2 hadthe highest ending ethanol titer in the test that began with addedethanol and was chosen for isolation of single cell derived strains. Thestrain isolation procedure described for the screen that derived strain12-18X-2-36 was run twice and the results of both rounds of screeningare shown in Tables 4 and 5. Screens were started in HYAc/YE medium withno added ethanol, or with 40 g/L added ethanol.

TABLE 4 Analysis of isolated pure cultures from week 2 of the turbidosatadaptation of 12-18X-2-36 grown in 12 ml HYAc/YE or HYAc/YE + ethanolfermentations (round 1) HYAc/YE with no added ethanol HYAc/YE with 40g/L ethanol remaining Remaining Remaining Remaining Sampling glucosexylose ethanol glucose xylose ethanol culture time OD 600 nm (g/L) (g/L)(g/L) OD 600 nm (g/L) (g/L) (g/L) ZW705 0.0 0.5 77.7 50.9 0.0 0.50 75.149.2 28.1 24.0 0.8 56.5 50.2 9.0 0.42 69.8 50.0 24.5 48.0 1.8 12.6 41.628.2 0.75 62.6 49.6 27.5 72.0 1.9 3.8 24.0 46.7 0.60 52.7 48.7 36.5 1-320.0 0.5 77.7 50.9 0.0 0.50 75.1 49.2 28.1 24.0 0.8 55.3 49.6 12.0 0.4165.3 48.0 35.2 48.0 1.9 7.8 38.5 39.1 0.68 57.3 47.6 38.2 72.0 2.4 2.621.2 51.6 0.72 50.8 47.5 41.0 1-44 0.0 0.5 77.7 50.9 0.0 0.50 75.1 49.228.1 24.0 0.8 53.7 49.9 12.6 0.44 66.7 48.2 34.7 48.0 1.9 6.5 37.5 40.10.75 60.8 48.5 35.5 72.0 2.2 1.8 21.7 50.1 0.65 55.8 48.1 37.4 2-24 0.00.5 77.7 50.9 0.0 0.50 75.1 49.2 28.1 24.0 0.7 59.4 49.7 9.8 0.41 67.648.6 32.0 48.0 1.9 4.8 34.9 42.8 0.69 61.2 48.6 34.2 72.0 2.3 2.5 18.651.3 0.64 55.9 48.6 36.9 2-47 0.0 0.5 77.7 50.9 0.0 0.50 75.1 49.2 28.124.0 0.9 52.1 49.5 13.5 0.49 66.3 48.7 30.8 48.0 1.8 9.6 40.3 37.0 0.9058.6 48.8 34.4 72.0 1.8 2.7 27.6 46.3 0.61 51.8 48.4 38.2 3-8  0.0 0.577.7 50.9 0.0 0.50 75.1 49.2 28.1 24.0 0.8 56.9 49.8 10.9 0.44 66.6 48.033.8 48.0 2.0 50.0 34.3 43.0 0.76 58.3 47.9 37.2 72.0 2.4 2.6 19.6 50.70.85 50.6 47.7 40.8 3-45 0.0 0.5 77.7 50.9 0.0 0.50 75.1 49.2 28.1 24.00.6 60.4 950.0 9.2 0.44 67.6 48.4 34.3 48.0 1.3 14.1 43.4 33.6 0.55 61.448.1 36.7 72.0 1.8 2.6 19.6 45.1 0.52 57.2 47.9 38.0 4-17 0.0 0.5 77.750.9 0.0 0.50 75.1 49.2 28.1 24.0 0.6 58.8 49.6 10.0 0.44 66.7 48.3 32.848.0 1.4 5.7 38.0 40.4 0.60 59.7 48.4 34.8 72.0 1.9 2.4 29.6 49.6 0.5455.2 48.6 35.5 4-27 0.0 0.5 77.7 50.9 0.0 0.50 75.1 49.2 28.1 24.0 0.849.8 49.4 14.6 0.52 63.6 48.4 33.7 48.0 1.6 4.3 34.1 44.0 0.74 53.8 48.237.9 72.0 2.1 2.6 23.7 48.1 0.68 47.5 48.1 40.3 5-6  0.0 0.5 77.7 50.90.0 0.50 75.1 49.2 28.1 24.0 0.6 53.9 79.7 12.6 0.46 59.7 48.7 35.5 48.01.8 5.8 36.9 41.9 0.90 47.7 48.8 40.3 72.0 1.9 2.7 23.8 48.7 0.59 41.548.6 42.9 5-35 0.0 0.5 77.7 50.9 0.0 0.50 75.1 49.2 28.1 24.0 0.8 52.749.7 13.1 0.42 65.8 48.2 33.3 48.0 1.7 5.7 36.8 41.6 0.74 58.4 48.0 36.372.0 1.9 2.4 22.7 49.6 0.80 53.6 48.1 38.1

When starting without added ethanol most strains used all of the glucosein the hydrolysate medium but did not use all of the xylose, so thattotal ethanol production was dependent on the extent of xyloseutilization. Several strains used more xylose and produced more ethanolthan did the non-adapted parent strain ZW705. When 40 g/L ethanol wasadded to the starting medium, growth and sugar utilization was much lessin all strains. In this test xylose use was very low or xylose was notconsumed at all in most cases. Three strains used significantly moreglucose and produced more ethanol than ZW705. The best of those was thestrain Adapted 5-6, shown in Table 4.

TABLE 5 Analysis of isolated pure cultures from week 2 of the turbidosatadaptation of 12-18X-2-36 grown in 12 ml HYAc/YE or HYAc/YE + ethanolfermentations (round 2) HYAc/YE with no added ethanol HYAc/YE with 40g/L ethanol remaining remaining remaining remaining Sampling OD glucosexylose ethanol OD glucose xylose ethanol culture time 600 nm (g/L) (g/L)(g/L) 600 nm (g/L) (g/L) (g/L) ZW705 0.0 0.5 77.8 51.5 0.0 0.50 74.949.1 30.6 24.0 0.9 40.7 48.6 19.0 0.60 56.2 49.0 34.0 48.0 2.5 2.9 26.548.3 1.14 28.4 46.9 48.8 72.0 2.9 2.2 17.9 52.7 1.49 17.5 44.7 55.3 6-40.0 0.5 77.8 51.5 0.0 0.50 74.9 49.1 30.6 24.0 0.9 19.0 46.0 30.6 0.7954.4 49.0 34.9 48.0 2.4 2.9 28.4 47.2 1.75 18.9 44.2 55.2 72.0 2.6 2.118.4 51.8 1.98 9.2 39.8 61.7  6-43 0.0 0.5 77.8 51.5 0.0 0.50 74.9 49.130.6 24.0 1.3 28.0 47.7 25.0 0.52 58.6 48.3 36.5 48.0 2.7 2.7 20.9 50.50.65 44.9 47.6 44.0 72.0 2.8 2.2 14.6 52.9 1.12 37.9 47.3 46.8  7-13 0.00.5 77.8 51.5 0.0 0.50 74.9 49.1 30.6 24.0 1.6 17.5 46.6 29.7 0.51 57.048.2 38.0 48.0 32.7 2.6 18.2 50.6 0.99 35.7 46.3 49.4 72.0 3.3 2.2 11.853.3 1.99 25.9 44.9 53.8  7-31 0.0 0.5 77.8 51.5 0.0 0.50 74.9 49.1 30.624.0 1.4 23.1 46.9 27.7 0.77 39.5 48.0 39.5 48.0 2.5 2.7 19.2 52.3 1.3522.3 45.2 56.7 72.0 3.0 2.1 12.8 53.7 2.09 12.7 43.6 56.5 8-2 0.0 0.577.8 51.5 0.0 0.50 74.9 49.1 30.6 24.0 1.5 22.1 47.5 20.7 0.59 54.8 48.337.8 48.0 3.3 2.8 20.3 50.3 1.24 30.4 46.3 51.7 72.0 3.4 2.3 14.8 51.81.14 20.4 44.8 55.0  8-22 0.0 0.5 77.8 51.5 0.0 0.50 74.9 49.1 30.6 24.01.2 28.7 47.6 24.6 0.56 58.1 48.6 35.5 48.0 2.3 2.8 19.2 51.5 0.99 33.646.4 49.2 72.0 2.8 2.2 11.4 55.2 1.14 21.7 44.1 54.9 9-5 0.0 0.5 77.851.5 0.0 0.50 74.9 49.1 30.6 24.0 1.3 32.0 49.2 23.0 0.67 57.9 48.2 37.048.0 2.5 2.9 23.0 48.8 1.07 32.0 46.2 51.8 72.0 2.7 2.3 14.7 52.4 1.6822.2 44.3 55.6  9-21 0.0 0.5 77.8 51.5 0.0 0.50 74.9 49.1 30.6 24.0 1.428.4 48.0 24.9 0.51 58.7 48.3 35.9 48.0 2.6 2.7 21.1 50.4 0.88 36.4 47.048.4 72.0 2.7 2.3 13.4 53.5 1.06 26.1 45.3 52.2 10-17 0.0 0.5 77.8 51.50.0 0.50 74.9 49.1 30.6 24.0 0.9 42.1 49.4 18.4 0.32 62.8 48.4 33.9 48.02.4 3.0 24.7 49.0 0.43 51.7 47.8 41.2 72.0 2.8 2.3 14.0 52.7 0.55 45.647.7 41.0 10-32 0.0 0.5 77.8 51.5 0.0 0.50 74.9 49.1 30.6 24.0 1.1 37.648.9 20.1 0.50 59.3 48.4 34.1 48.0 2.3 2.8 23.4 47.3 1.05 35.2 46.9 46.672.0 2.4 2.3 15.7 51.0 1.34 22.6 45.0 52.3

Results were similar in the second strain isolation experiment. Severalstrains used more xylose than ZW705 in the no added ethanol test, andused more glucose in the added ethanol test. Many used more glucose inthe first 24 hours of growth and achieved higher cell mass as measuredby OD600 nm in the first 24 hours also. Of these, the strain Adapted7-31 was chosen for further testing.

Example 3 Performance Testing of Hydrolysate Adapted Strains

Seed Fermentation

Seed fermentation was performed in a 1 L fermenter (Sartorious StedimBIOSTAT). Sterilized, empty fermenters were filled withfilter-sterilized halfYEMaxSM. Seed fermentations were performed at 33°C. and pH 5.5, using unfiltered 4 N NH₄OH as the base to control to pH5.5. Seed fermentations were inoculated with sufficient volume of frozenstock cells that were grown for about 7.5 hr to OD600 of about 2.5, inhalfYEMaxSM. Cells were diluted into the 1L fermenter to give a startingOD600 nm of about 0.025. In general, seed fermentations were harvestedwhen ˜120 g/L glucose had been consumed and/or OD600 of about 10 hadbeen reached. The seed fermentations were periodically sampled tomonitor growth, and harvested at about 18.5 hr.

Hydrolysate Fermentation

Hydrolysate fermentation was performed in a 1 L fermentor (SartoriousStedim BIOSTAT). Sterilized, empty fermentors were filled with 450 ml ofcorn cob hydrolysate prepared as described in General Methods.Hydrolysate fermentations were performed at pH 5.8 using unfiltered 4 NNaOH as the base to adjust pH. Hydrolysate fermentation began at 33° C.Hydrolysate fermentations were inoculated with 10 volume % (50 ml) ofseed from the seed fermentation (see above) to generate an initial OD ofabout 1.0. The hydrolysate fermentations were periodically sampled tomonitor reaction progress. The samples were assayed for glucose, xyloseand ethanol as described in General Methods.

Two hydrolysate fermentations were run one week apart, each with thecontrol strain ZW705 and the selected strain from adaptation, Adapted7-31 (Example 2). The time course of the fermentations is shown inFIG. 1. Adapted 7-31 used glucose faster than ZW705 and used more totalxylose to achieve a higher ethanol titer during the course of thefermentation.

Hydrolysate fermentations were run and assayed as described above tocompare the control strain ZW705, strain Adapted 7-31, and strainAdapted 5-6 (see Example 2). Results are shown in FIG. 2. Strain Adapted5-6 performance was equivalent to that of strain Adapted 7-31. Both ofthese adapted strains achieved a higher final ethanol titer by usingglucose more rapidly at the beginning of the fermentation and by usingmore xylose by the end of the fermentation.

Example 4 Comparative DNA Sequencing of Adapted Strains

The whole genome sequence of wild type Zymomonas mobilis (ZM4) has beendescribed (Jeong-Sun Seo et. al, Nature Biotechnology. 23, 2005). A wildtype starting strain (ZW1; ATCC 31821), an intermediate strain (ZW658;ATCC PTA-7858) and ZW705, were sequenced using high throughput 454technology (Shendure and Ji, Nature Biotech. 26:1135 (2008)) andcompared to the published wild type sequence. Strains Adapted 5-6 andAdapted 7-31 were sequenced using Ilumina technology (Shendure and Ji,Nature Biotech 26:1135 (2008)). From the wild type sequence, thesequence of ZW658 and the separately determined insertion sites of theintentional sequence changes coming from insertion of the two xyloseutilization operons and the knockout of the GFOR gene (described inGeneral Methods), a consensus sequence was made to which sequences fromadapted strains could be compared. The description of sequencing andgenome assembly follows.

Sequencing

Strains were sequenced using the sequencing technologies Illumina/Solexaand Roche-454. These massively parallel sequencing methods give veryhigh throughput of short reads. In the case of Illumina, it gives morethan 200 million reads that are 100 bp long. For Roche-454 outputs 1million reads are 500 bp long. Both methods allow sequencing from bothends of genomic DNA fragments to produce paired-end reads

Hundreds of millions of short reads from Adapted 5-6 and Adapted 7-31strains were aligned to a reference genome sequence prepared from thewild type, ZW658, and ZW705 sequences. The resulting alignment wasanalyzed to collect information on coverage and variations. Since manyreads can align to a specific region, the alignment is a pile up ofreads against the reference and the depth coverage of a given positionis the number of reads that cover that position. If at a position theconsensus base is different from the reference base, the position issaid to have a Single Nucleotide Polymorphism (SNP) variation.

In the comparison of the Adapted 5-6 and Adapted 7-31 sequences to thestarting strain for adaptation ZW705 sequence, a single base change, orSNP, was identified for each adapted strain. The SNP for Adapted 5-6 wasat position 1453116 which is in the open reading frame of a genedesignated zmo1330. The SNP for Adapted 7-31 was in the same openreading frame, but the change was at a different position (1453863). Thechange in Adapted 5-6 is in position 1097 of the coding region (SEQ IDNO:1) and is a change from C to G at that position. This mutationresults in a codon change for amino acid 366 from ACA to AGA, resultingin a change in amino acid 366 from threonine to arginine in the encodedprotein. The change in Adapted 7-31 is in position 350 of the codingregion (SEQ ID NO:1) and is a change from C to T at that position. Thismutation results in a codon change for amino acid 117 from TCT to TTTresulting in a change in amino acid 117 from serine to phenylalanine.

The protein encoded by zmo1330 was used in a BLAST of the transporterclassification data base (Saier Lab Bioinformatics Group; Saier et al.(2009), Nucl. Acids Res., 37: D274-8) which identified the protein asbelonging to the aaeB family. This family of proteins is characterizedby having a hydrophobic N terminus that predicts 5 to 6 membranecrosses, a fairly long and less hydrophobic middle section, and then asimilar 5 to 6 membrane crosses in the C-terminus. This result suggestedthat the zmo1330 encoded protein is a membrane transport protein.

In the published Z. mobilis genome sequence (Seo et al., ibid; NCBIReference: NC_(—)006526.2), zmo1330 corresponds to zmo1432 which isannotated as encoding a “fusaric acid resistance protein”. The set ofproteins required for resistance to fusaric acid in Pseudomonas cepaciawas identified by Utsumi et al. (Agric. Biol. Chem. 55:1919-1918(1991)). Among the set of open reading frames (ORFS) in what appeared tobe one operon conferring fusaric acid resistance is one designated fusBby Utsumi et al. (ibid) The sequence of the protein encoded by fusB isidentical to Bcenm03_(—)1426 from Burkholderia cenocepacia (SEQ IDNO:7), which was aligned with the protein encoded by zmo1432. The twoprotein sequences align with 3 small gaps and are 22% identical, but 63%similar (Clustal W alignment) as shown in FIG. 3. The sequence ofBcenm03_(—)1426 has about the same identity and similarity to thesequence of AaeB (SEQ ID NO:8), which is the larger of two proteinsencoded in an operon in E. coli that were shown to be required fortolerance to p-aminobenzoic acid (pABA) (VanDyk et al J. Bact.186:7196-7204 (2004)). Zmo1432 is 17% identical and 55% similar to E.coli aaeB. Alignment of proteins encoded by zmo1432 and to aaeB is shownin FIG. 4.

What is claimed is:
 1. A recombinant, xylose-utilizing,ethanol-producing microorganism of the genus Zymomonas, having at leastone genetic modification in the zmo1432 open reading frame, wherein saidzmo1432 open reading frame having at least one genetic modificationencodes a polypeptide having an amino acid sequence with at least about95% identity to the amino acid sequence as set forth in SEQ ID NO:2,wherein the presence of the mutant protein increases ethanol productionby the Zymomonas during fermentation in biomass hydrolysate medium ascompared to production by a Zymomonas lacking the genetic modificationunder the same conditions.
 2. The recombinant Zymomonas of claim 1wherein the genetic modification is a mutation in the zmo1432 codingregion that results in an amino acid substitution selected from thegroup consisting of: 1) a substitution at position 366 in SEQ ID NO: 2of arginine for threonine; and 2) a substitution at position 117 in SEQID NO: 2 of phenylalanine for serine.
 3. The recombinant Zymomonas ofclaim 1 having a higher tolerance to biomass hydrolysate as comparedwith a Zymomonas lacking the at least one genetic modification in thezmo1432 open reading frame.
 4. A polynucleotide encoding a proteinhaving the amino acid sequence selected from the group consisting of SEQID NO:3 and SEQ ID NO:
 4. 5. A method for the production of arecombinant Zymonomas ethanologen comprising: a) providing axylose-utilizing, ethanol-producing microorganism of the genus Zymomonascomprising a zmo1432 open reading frame encoding a polypeptide havingthe amino acid sequence as set forth in SEQ ID NO: 2; and b) introducinga mutation in the zmo1432 open reading frame of a) such that expressionof the mutated open reading frame expresses a polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NO:3and SEQ ID NO:4.
 6. A method of the production of ethanol comprising: a)providing the recombinant Zymomonas of claim 1; b) providing a biomasshydrolysate medium comprising xylose; and c) growing the Zymomonas of a)in the biomass hydrolysate medium of b) wherein ethanol is produced.